Highly efficient production of L-valine by multiplex metabolic engineering of Corynebacterium glutamicum / 生物工程学报

As a branched chain amino acid, L-valine is widely used in the medicine and feed sectors. In this study, a microbial cell factory for efficient production of L-valine was constructed by combining various metabolic engineering strategies. First, precursor supply for L-valine biosynthesis was enhanced by strengthening the glycolysis pathway and weakening the metabolic pathway of by-products. Subsequently, the key enzyme in the L-valine synthesis pathway, acetylhydroxylate synthase, was engineered by site-directed mutation to relieve the feedback inhibition of the engineered strain. Moreover, promoter engineering was used to optimize the gene expression level of key enzymes in L-valine biosynthetic pathway. Furthermore, cofactor engineering was adopted to change the cofactor preference of acetohydroxyacid isomeroreductase and branched-chain amino acid aminotransferase from NADPH to NADH. The engineered strain C. glutamicum K020 showed a significant increase in L-valine titer, yield and productivity in 5 L fed-batch bioreactor, up to 110 g/L, 0.51 g/g and 2.29 g/(L‧h), respectively.

Re-investigation after 8 years on the publication of Chinese clinical trials after registration / 中华医学科研管理杂志

Objective To investigate the publication status and quality in registration centers and journals of Chinese clinical trials after registration.Methods The Chinese clinical trials registered on the US Clinical Trial Registration website (www.clinicaltrials.gov) before July 24,2009 were searched.China Knowledge Network (CNKI),Wanfang Database,VIP Database,PubMed,and EMbase Database were searched by computer from inception to December 31,2017,to investigate the outcome publication of these clinical trials in the registration center and journals.Results A total number of 654 clinical trials were included in this study.Only 25 clinical trials,accounted for 3.8％,published trial outcome on the US Clinical Trials Register,74 clinical trials,accounted for 11.3％,were published in the journals (effective literature).Most of the trials took 2 to 3 and in both from registration to completion and study closure to outcome publication.Conclusions The proportion and quali ty of clinical trial outcome publication in China were low,which alerting us that researcher should pay more attention to the trial outcome publication.

Optimization of Purification Technology of N-（E）-p-coumaroyltyrosine in Leaves of Abrus cantoniensis / 中国药房

OBJECTIVE： To establish a method for content determination of N-（E）-p-coumaroyltyrosine in leaves of Abrus cantoniensis， and to optimize its purification technology. METHODS： HPLC method was adopted for the content determination of N-（E）-p-coumaroyltyrosine in A. cantoniensis. The determination was performed on Hypersil BDS C18 column with mobile phase consisted of 0.1％ formic acid water （A）-methanol （B） （gradient elution） at a flow rate of 1.0 mL/min. The column temperature was set at 25 ℃. The detection wavelength was set at 300 nm， and sample size was 10 μL. Using polyamide resin as material， the yield of N-（E）-p-coumaroyltyrosine as indicators， single factor test was used to optimize the purification technology of N-（E）-p-coumaroyltyrosine， such as concentration， sample size， stationary adsorption time. RESULTS： The linear range was 2.575-51.50 μg （r＝0.999 9） for N-（E）-p-coumaroyl-tyrosine. The limit of quantification was 0.000 618 μg， and the detection limit was 0.000 129 μg. RSDs of precision， stability and reproducibility tests were all lower than 3％. The recoveries were 97.04％-102.43％ （RSD＝2.06％， n＝6）. The optimal purification technology was as follows： the concentration of the sample solution was 0.04 g /mL （by the leaves of A. cantoniensis）； sample capacity 50 mL； the sample flow rate was 1.0 mL/min； the stationary adsorption time was 20 min； the eluting solvents were ammonia containing water （containing 0.1％ acetic acid）， 20％ ethanol （containing 0.1％ acetic acid） and ammonia（pH 10）. Average yield was 98.94％，average dry paste content was 61.17 mg/g， and average dry paste purity was 19.73％ by optimal purification technology. CONCLUSIONS： Established method is simple， accurate and stable. The optimized technology is stable and feasible.

Protective effect of hydrogen-rich water on oxidative stress cell model and the impact of the phosphatidylinositol 3 kinase/protein kinase B pathway / 中华危重病急救医学

Objective To explore the protective effect of hydrogen-rich water on the oxidative stress injury of astrocytes in mice and its effect on phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signal pathway. Methods In vitro, mice astrocytes were cultured and the logarithmic growth period cells were taken for experiment. ① Experiment one: some cells were acted by 1.25, 2.50, 5.00, 10.00 μmol/L hydrogen peroxide (H2O2) for 20 minutes to determine the appropriate concentration required for astrocyte damage induced by H2O2; cultivating 3, 6, 9, and 12 hours with hydrogen-rich water of 25, 50, 100, and 200 μmol/L, respectively, to determine the concentration and time of hydrogen-rich water pretreatment; the 50 μmol/L hydrogen-rich water was cultured together with PI3K/Akt signal pathway inhibitors wortmannin (WM) 200 nmol/L or 400 nmol/L to determine the best inhibition concentration of wortmannin. Astrocyte activity was detected by methyl thiazolyl tetrazolium (MTT) colorimetry.② Experiment two: some cells were divided into blank control group, H2O2 injury group, hydrogen-rich water pretreatment group (HW+H2O2 group), and co-culture of hydrogen-rich water and wortmannin pretreatment group (HW+WM+H2O2 group). The mRNA expressions of PI3K and Akt were detected by reverse transcription-polymerase chain reaction (RT-PCR); the protein expressions of PI3K, Akt and phosphorylated Akt (p-Akt) were detected by Western Blot. Results ① Experiment one: the survival rate of the blank control group was 100%. Cell activity gradually decreased with the increase of H2O2 concentration, and the survival rate of the H2O2 action 20 minutes cells of 2.50 μmol/L was reduced to about 50%, so a cell injury model was established at this concentration. With the increase of hydrogen-rich water pretreatment concentration, and the duration of action, the cell survival rate increased first and then decreased. The cell survival rate was highest when 50 μmol/L hydrogen-rich water was pretreated with 9 hours, so a hydrogen-rich water pre-protection model was established. After 200 nmol/L or 400 nmol/L wortmannin was cultured together with hydrogen-rich water, cell activity was inhibited, and the cell survival rate of 200 nmol/L wortmannin group was no significantly different compared with that of H2O2 injury group, so the astrocyte suppression model was established. ② Experiment two: compared with the blank control group, the mRNA expressions of PI3K and Akt and the protein expressions of PI3K, Akt and p-Akt were significantly decreased in the H2O2 injury group. Compared with the H2O2 injury group, the PI3K, Akt mRNA expressions and PI3K, Akt, p-Akt protein expressions were significantly increased in the HW+H2O2 group [PI3K mRNA (2-ΔΔCT): 0.843±0.019 vs. 0.631±0.038, Akt mRNA (2-ΔΔCT): 0.591±0.025 vs. 0.558±0.037, PI3K/β-actin: 1.277±0.008 vs. 0.757±0.004, Akt/β-actin: 1.308±0.015 vs. 0.682±0.006, p-Akt/β-actin: 1.210±0.005 vs. 0.614±0.005, all P < 0.05]. The mRNA expressions of PI3K, Akt in the HW+WM+H2O2 group was 0.784±0.159 and 0.556±0.037, respectively, and the protein expressions of PI3K, Akt, p-Akt was 0.715±0.006, 0.686±0.005, and 0.606±0.004, respectively, both were significantly lower than those in HW+H2O2 group (all P < 0.05), and there was no significant difference with H2O2 injury group (all P >0.05). Conclusion Hydrogen-rich water activates the PI3K/Akt pathway, thereby mediates mice astrocytes to exert the biological function of antioxidant.